ANTIANXIETY PROPERTY OF TECOMA STANS LINN LEAVES EXTRACT IN EXPERIMENTAL ANIMALS - METHODOLOGY

 

4. METHODOLOGY

 

4.1 MATERIALS

Drug: scopolamine (Shreeji Pharma International Pvt. Ltd), Phenobarbitol (Ranbaxy), Pentobarbitol (Sun Pharma.Pvt.Ltd.), Diazepam (Reliance).

 

Plant: The Tecoma stans leaves are found throughout India. The description, history, cultivation and constituents of the plant have been already described. The plant was authenticated by Scientist J. Jayanthi, Botanical Survey of India. Pune, Maharashtra (Annexure -I).

 

Reagents

• Benedict's reagent.
• Barfoed's reagent.
• Million's reagent.
• Dragendroff's reagent.
• Hagers reagent.
• Mayer's reagent.
• Wagner's reagent.

 

Chemicals

• All chemicals used were of analytical grade.
• Petroleum ether-SDFCL.Fine-chem., Ltd, Mumbai.
• Chloroform- SDFCL.Fine-chem., Ltd, Mumbai.
• Ethanol- Ghangshu Yangyuan Chemical China.

4.1.1 Instrument:

• Elevated plus maze apparatus- INCO, Medicraft. India.
• Open field test apparatus.
• Light/dark test box.
• Hole board test apparatus
• Soxhlet apparatus- Biotechnics, India.
• Double distillatory- Infustil India Pvt. Ltd. Bangalore.
• Electronic weighing balance- Citizen Scale, India.
• Oral feeding needle- Space Lab, Nasik.

 

4.1.2 Animals:

            Albino male wistar rats and albino mice weighing between 150 to 250g and 20 to 30 gm respectively were procured form registered breeders (Mahavir Enterprise, Hyderabad).The animals were housed under standard conditions of temperature 20 ± 25 ⁰C and relative humidity 30- 70% with a 12:12 light-dark cycle. The animals were fed with standard pellet diet and water ad libitum. Approval of CPCSEA for the Institutional Animal Ethics Committee (IAEC) no. 346/CPCSEA of Luqman College of Pharmacy, Gulbarga was taken for conducting anxiolytic activity.

 

4.2. Methodology:

4.2.1. Extraction^{152, 153, 154}:-

            The plants collected were carefully protected and leaves were separated. The leaves were carefully washed with tap water and left to dry for 20 days in the shade at room temperature. Then they were stored in well sealed cellophane bags, so as to prevent from the environmental effects.

            The shade dried powdered leaves were used for the extraction with Petroleum ether, Chloroform, Ethanol and Distilled water. In each case, the powder weighing approximately 325-350 gm was extracted by adding 1000 ml of the solvents. The duration of extraction varies with the solvent and was found to be between 08-12 h with all the solvents. The extract was filtered and the filtrate evaporated to dryness under reduced pressure using a rotatory evaporator.

 

4.2.2. Qualitative chemical test^{155, 156}:

Preliminary phytochemical investigation of extract:-

            Qualitative chemical tests were conducted for petroleum ether, chloroform, ethanolic and aqueous extracts of leaves of Tecoma stan linn to identify the various phytoconstituents. The various tests and reagents used are given below and observations are recorded and tabulated in results (Table 1 and 2).

 

Tests for Carbohydrates:

Molisch's test (General test):

            To 2-3 ml aqueous extract, few drops of α-naphthol solution in alcohol was added, shaken and concentrated H2SO4 was added from the sides of the test tube. It was observed for violet ring at the junction of two liquids.

 

For Reducing Sugars:

a) Fehling's test:

            1 ml Fehling's A and 1ml Fehling's B solutions was mixed and boiled for one min. Equal volume of test solution was added. Heated in boiling water bath for 5-10 min and observed for a yellow, then brick red precipitate.

b) Benedict's test:

            Equal volume of Benedict's reagent and test solution in test tube were mixed. Heated in boiling water bath for 5 min. Solution may appear green, yellow or red depending on amount of reducing sugar present in test solution.

 

Test for Monosaccharide’s:

Barfoed's test:

            Equal volumes of Barfoed's reagent and test solution were added. Heated for 1-2 min, in boiling water bath and cooled. Observe for red precipitate.

 

Test for Hexose Sugars:

            Cobalt-chloride test: 3 ml of test solution was mixed with 2ml cobalt chloride, boiled and cooled, added few drops of FeCl3 and NaOH solution. Solution was observed for greenish blue (glucose), purplish (Fructose) or upper layer greenish blue and lower layer purplish (Mixture of glucose and fructose).

 

Tests for Non-Reducing Sugars:

a)      Test solution does not give response to Fehling's and Benedict's test.

b)      Tannic acid test for starch: With 20% tannic acid, test solution was observed for precipitate.

 

Tests for Proteins:

a) Biuret test:

            (General test): To 3 ml test solution added 4% NaOH and few drops of 1% CUSO4 solution and observed for violet or pink color.

b) Millon's test: (for proteins):

            Mixed 3 ml test solution with 5 ml Millon’s reagent, white precipitate obtained. Precipitate warmed turns brick red or precipitate dissolves giving red color.

 

c) Xanthoprotein test: (For protein containing tyrosine or tryptophan):

            Mixed 3ml test solution with 1 ml concentrated H2SO4, observed for white precipitate.

 

d) Test for protein containing sulphur:

            Mixed 5 ml test solution with 2 ml 40% NaOH and 2 drops 10% lead acetate solution. Solution was boiled, turns black or brownish due to lead sulfide formation.

 

Tests for Steroids:

a) Salkowski Reaction:

            To 2 ml of extract, 2 ml chloroform and 2 ml concentrated H2SO4 was added. Shook well, whether chloroform layer appeared red and acid layer showed greenish yellow fluorescence was observed.

 

b) Liebermann-Burchard Reaction:

            Mixed 2ml extract with chloroform. Added 1-2ml acetic anhydride and 2 drops concentrated H2SO4 from the side of test tube, observed for first red, then blue and finally green color.

 

c) Liebermann’s reaction:

            Mixed 3 ml extract with 3 ml acetic anhydride. Heated and cooled. Added few drops concentrated H2SO4, observed for blue color.

Tests for Amino Acids:

a) Ninhydrin test (General test):

            3 ml test solution and 3 drops 5% Ninhydrin solution were heated in boiling water bath for 10 min and observed for purple or bluish color.

 

b) Test for Tyrosine:

            Heat 3 ml test solution and 3 drops Millon’s reagent. Solution was observed for dark red color.

 

c) Test for Tryptophan:

            To 3 ml test solution added few drops glycoxalic acid and concentrated H2SO4 observed for reddish violet ring at junction of the two layers.

 

Tests for Flavonoids:

a)      Shinoda test: - To dried powder or extract, added 5 ml 95% ethanol, few drops concentrated HCl and 0.5 g magnesium turnings. Pink color was observed.

b)      To small quantity of residue, added lead acetate solution observed for Yellow colored precipitate.

c)      Addition of increasing amount of sodium hydroxide to the residue was observed as to whether it showed yellow coloration, which was decolorized after addition of acid.

d)     Ferric chloride test: To test solution, added few drops of ferric chloride solution observed for intense green color.

 

Tests for Alkaloids:

a) Dragendroff's test:

            To 2-3 ml filtrate added few drops Dragendroff's reagent and was observed for orange brown precipitate.

 

b) Mayer's test:

            2-3 ml filtrate with few drops Mayer's reagent was observed for precipitate.

 

c) Wagner's test:-

            2-3 ml filtrate with few drops of Wagner's reagent was observed for reddish brown precipitate.

 

Tests for Tannins and Phenolic Compounds:

            To 2-3 ml test solution, added few drops of following solutions and was looked for respective coloration or precipitate:

a)      5% Ferric chloride solution: - Deep blue-black colored.

b)      Lead acetate solution: - White precipitate.

c)      Bromine water: - Decoloration of bromine water.

d)     Acetic acid solution: - Red color solution.

e)      Potassium dichromate: - Red precipitate.

f)       Dilute iodine solution: - Transient red color.

g)      Dilute Nitric acid: - Reddish to yellow color.

 

Tests for Glycosides:

General test for Glycosides:

Part A:

            To 2-3 ml of extract dil. H2SO4 was added and heated on a water bath for 1- 2 min. Neutralize with 10% NaOH, check with litmus paper and to resulting solution add Fehling's A and B. Increased red precipitate in this case shows glycosides are present.

 

Part B:

            To 2-3 ml of extract, water was added and heated. According to need, NaOH was added for neutralization and also added equal quantity of water. To the resulting solution added Fehling's A and B. Increased red precipitate in this case showed glycosides are absent. Compare Part A and B.

 

Tests for Cardiac Glycosides:

a) Baljet's test:-

            The test solution was observed for yellow to orange color with sodium picrate.

 

b) Legal's test (For cardenoloids):

            To aqueous or alcoholic test solution, added 1 ml pyridine and 1 ml sodium nitroprusside, observed for pink to red color.

 

c) Test for deoxysugars (Keller Killani test):

            To 2 ml extract added glacial acetic acid, one drop of 5% FeCl3 and concentrated H2SO4, observed for reddish brown color at junction of the two liquid and upper layers bluish green.

d) Liebermann’s test (For bufadenolids):

            Mixed 3 ml extract with 3 ml acetic anhydride. Heated and cooled. Added few drops concentrated H2SO4 observed for blue color.

 

Tests for Saponin Glycosides:

a) Foam test:

            The drug extract or dry powder was shaken vigorously with water. Persistent foam was observed.

 

4.3. Methods:

4.3.1. Acute oral toxicity study¹⁵⁷:

            Acute oral toxicity study for the formulation was carried out using OECD guideline 425 (Adopted 3^rd October 2008). The test procedure minimizes the number of animals required to estimate the oral acute toxicity of a chemical and in addition estimation of LD50, confidence intervals. The test also allows the observation of signs of toxicity and can also be used to identify chemicals that are likely to have low toxicity.

 

Principle of the FDP:

            The fixed dose procedure is method for assessing acute oral toxicity that involve the identification of a dose level that cause evidence of non-lethal toxicity(termed evident toxicity) rather than a dose level that cause lethality. Evident toxicity is terms describing clear signs of toxicity following administration of test substance, such that an increase to the next highest fixed dose would result in the development of sever toxic signs and probably mortality.

Procedure:

     As suggested, after acclimatization of animals for 4-5 days, study was carried out as follows:

• Healthy, young adult Albino Swiss female mice (20-30gm), nulliporous and non pregnant were used for this study Food, but not water was with held for 3-4 hours and further 1-2 hours post administration of sample under study.
• Fixed dose level of 5, 50, 500 mg/kg were initially chosen as dose level that would be expected to allow the identification of dose producing evident toxicity.
• During the validation procedure, a fixed dose of 2000mg/kg was added to provide more information on substance of low acute toxicity.
• Dosed one animal at the test dose by oral route.
• Since, this first test animal survived, four other animals were dosed (orally) as subsequent days, so that a total of five animals were tested.

 

Observation:

            After the administration of aqueous and ethanolic extracts, animals were observed individually during the first 30 min and periodically during 24 hours with special attention during the first four hours and daily thereafter for a period of 14 days. Once daily animals were observed principally in relation to changes in skin, fur, eyes and mucous membrane (nasal) and also autonomic symptoms like sedation, lacrimation, perspiration, piloerection, urinary incontinence and control nervous system (ptosis, drowsiness, gait tremors and convulsion).

 

4.3.2. Elevated Plus-Maze Test in mice^{131,158,159,160} :

            Elevated plus maze (EPM) one of the commonly used animal model (exteroceptive aversion stimulus model) for testing antianxiety drugs was employed to assess anxiolytic activity of sample under study. EPM is based on the apparent natural aversion of rodents to open and high spaces animals have tendency to spend more time in enclosed arms than in the open arms.

 

Procedure:

            Albino mice of either sex weighing between 20-30 gm were dividing into four experimental groups of six animals each.

       Group I - Control (2% gum acacia).

       Group II- Standard drug (Diazepam 2mg/kg p.o)

       Group III - Aqueous extracts dose of AE1 (400 mg/kg p.o).

       Group IV - Aqueous extracts dose of AE2 (800 mg/kg p.o).

       Group V - Ethanolic extracts dose of EE1 (400 mg/kg p.o).

       Group VI - Ethanolic extracts dose of EE2 (800 mg/kg p.o).

            Standard drug (Diazepam) was administered 45 min prior to testing and extracts were administered p.o 45 min prior to testing. Anxiolytic activity was measured using the elevated plus maze test. The maze consisted of two open (28 cm x 5 cm) and two closed (28 cm x 5 cm x 14 cm) arms, extending from the central platform (5 cm x 5 cm) and elevated up to the height of 25 cm above the floor. The entire maze was made of clear Plexiglass. Mice were individually placed on the centre of the maze facing an open arm, and the number of entries and the time spend in closed and open arm were recorded during a 5 min observation period. Arm entries were defined as entry of all four paws in to the arm.

The following parameters were calculated for each animal.

a)      % of open arm time

b)      % of close arm time

c)      % open arm entry

d)     % closed arm entry

4.3.3. Hole-board test in rats¹⁶¹:

            Albino rats of either sex weighing between 150-250 gm were dividing into four groups of six animals each. Standard drug (Diazepam) was administered 30 min prior to testing and extracts were administered p.o 45 min prior to testing.

            Group I - Control (2% gum acacia).

            Group II- Standard drug (Diazepam 2mg/kg p.o)

            Group III - Aqueous extracts dose of AE1 (400 mg/kg p.o).

            Group IV - Aqueous extracts dose of AE2 (800 mg/kg p.o).

            Group V - Ethanolic extracts dose of EE1 (400 mg/kg p.o).

            Group VI - Ethanolic extracts dose of EE2 (800 mg/kg p.o).

Procedure:

            The hole-board apparatus was an open-field arena with 16 equally spaced holes of 3 cm diameter in the floor, similar to the box used in social interaction test. Rats were placed singly in the centre of the hole-board, and during a 5-min trial the following measures were recorded:

a)      Total time spend with the head dips

b)      Number of head dips in the holes

c)      Total locomotors activity (numbers of squares crossed)

d)     Number of rearing

            A head dip was scored if both eyes disappeared into the hole. The arenas were wiped with a damp cloth after each trial and any faeces removed.

 

4.3.4. Light-dark model transition test in mice^{162,163,164,165}:

           The light/dark transition test is based on the innate aversion of rodents to brightly illuminated areas and on the spontaneous exploratory behavior of rodent in response to mild stressors, that is, novel environment and light. A natural conflict situation occurs when an animal is exposed to an unfamiliar environment or novel objects. The conflict is between the tendency to explore and the initial tendency to avoid the unfamiliar (neophobia). The exploratory activity reflects the combined result of these tendencies in novel situations. Thus, in the light/dark test, drug induced increase in behavior in the white part of a two compartment box, in which a large white compartment is illuminated and a small black compartment is darkened, is suggested as an index of anxiolytic activity.

 

Procedure:

            Albino mice of either sex weighing between 18-25 gm were dividing into four groups of six animals each. Standard drug (Diazepam) was administered 30 min prior to testing and extracts were administered p.o 45 min prior to testing.

        Group I - Control (2% gum acacia).

       Group II- Standard drug (Diazepam 2mg/kg p.o)

       Group III - Aqueous extracts dose of AE1 (400 mg/kg p.o).

       Group IV - Aqueous extracts dose of AE2 (800 mg/kg p.o).

       Group V - Ethanolic extracts dose of EE1 (400 mg/kg p.o).

       Group VI - Ethanolic extracts dose of EE2 (800 mg/kg p.o).

            The apparatus for light/dark transition test consist of two compartments: one light area (27 L x 27 W x 27 H cm), illuminated by 100 W desk lamp was painted white, and the other dark area (18 L x 27 W x 27 H cm) was painted black. The two compartments were separated by a partition with a tunnel (7.5 x 7.5 cm) to allow passage from one compartment to the other. The experiments were performed between 09:00 and 14:00. Animal was placed in the centre of the light area with its back to the opening. The following parameters were recorded during 5 min:

a)      Total time spend in the illuminated part of the cage

b)      Time spend in the dark part of the cage

c)      Number crossing between the light and dark area

d)     Total number of crossings

 

4.3.5 Open field test^{: 167, 168}

Grouping:

      Six groups of six mice (20-30 gm) in each were used in this model.

            Group I - Control (2% gum acacia).

            Group II- Standard drug (Diazepam 2mg/kg p.o)

            Group III - Aqueous extracts dose of AE1 (400 mg/kg p.o).

            Group IV - Aqueous extracts dose of AE2 (800 mg/kg p.o).

            Group V - Ethanolic extracts dose of EE1 (400 mg/kg p.o).

            Group VI - Ethanolic extracts dose of EE2 (800 mg/kg p.o).

 

Procedure:

Standard drug (Diazepam) was administered 30 min prior to testing and extracts were administered p.o 30 min prior to testing.

           The apparatus consists of a wooden box (60X60X30cm). The apparatus is illuminated with 40-W lamp suspended 100cm above it. Mice will be fed orally with extract(s), vehicle (normalsaline) or diazepam (1mg/kg;p.o).

         

            After 30 minutes following parameters are observedand recorded during the test session of 5 minutes.

1.      Number of entry in central square.

2.      Time spent in Central Square.

3.      Number of squares crossed.

4.      Number of assisted rearing.